Histidine-containing Dipeptides Research Articles

Carnosine/histidine-containing dipeptide supplementation improves depression and quality of life: systematic review and meta-analysis of randomized controlled trials.

Mental ill-health is a common and growing issue, affecting 1 in 8 individuals or 970 million people worldwide in 2019. Histidine-containing dipeptides (HCDs) have been suggested to mitigate some aspects of mental ill-health, but a quantitative synthesis of the evidence is lacking. Therefore, a systematic review and meta-analysis of randomized controlled trials was conducted. To summarize the evidence on the effects of HCDs on mental health outcomes. A systematic literature search was performed using electronic databases (Medline via Ovid, Embase via Ovid, Scopus, Google Scholar, and Cochrane) from inception to October, 2022. Two authors independently extracted data using a structured extraction format. Data analysis was performed using STATA version 17. Random-effects models were used, and heterogeneity was assessed using the I2 test. Quality appraisal was performed using the Cochrane risk-of-bias 2.0 tool and the Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) approach. 5507 studies were identified, with 20 studies fulfilling the inclusion criteria. Eighteen studies comprising 776 participants were included in the meta-analysis. HCD supplementation (anserine/carnosine, l-carnosine, β-alanine) caused a significant reduction in depression scores measured with the Becks Depression Inventory (-0.79; 95% CI: -1.24, -0.35; moderate certainty on GRADE) when compared with placebo. An increase in quality-of-life scores measured with the 36-item Short-Form survey (SF-36) (0.65; 95% CI: 0.00, 1.30) and low certainty on GRADE in HCDs (anserine/carnosine, l-carnosine, β-alanine) when compared with placebo were found. However, the rest of the outcomes did not show a significant change between HCD supplementation and placebo. Although the number of studies included in the meta-analysis was modest, a significant mean reduction was observed in depression score as well as an increase in quality-of-life score for the HCD group when compared with placebo. Most of the studies included had small sample sizes with short follow-up periods and moderate to high risk of bias, highlighting the need for further, well-designed studies to improve the evidence base. PROSPERO registration no. CRD42017075354.

Genome-wide association studies of anserine and carnosine contents in the breast meat of Korean native chickens

Histidine-containing dipeptides (HCDs), such as anserine and carnosine, are enormously beneficial to human health and contribute to the meat flavor in chickens. Meat quality traits, including flavor, are polygenic traits with medium to high heritability. Polygenic traits can be improved through a better understanding of their genetic mechanisms. Genome-wide association studies (GWAS) constitute an effective genomic tool to identify the significant single-nucleotide polymorphisms (SNPs) and potential candidate genes related to various traits of interest in chickens. This study identified potential candidate genes influencing the anserine and carnosine contents in chicken meat through GWAS. We performed GWAS of anserine and carnosine using the Illumina chicken 60K SNP chip (Illumina Inc., San Diego, CA) in 637 Korean native chicken-red-brown line (KNC-R) birds consisting of 228 males and 409 females. The contents of anserine and carnosine in breast meat of KNC-R chickens were investigated. The mean value of the anserine and carnosine are 29.12 mM/g and 10.69 mM/g respectively. The genomic heritabilities were moderate (0.24) for anserine and high (0.43) for carnosine contents. Four and nine SNPs were significantly (P < 0.05) associated with anserine and carnosine, respectively. Based on the GWAS result, the 30.6 to 31.9 Mb region on chicken chromosome 7 was commonly associated with both anserine and carnosine. Through the functional annotation analysis, we identified HNMT and HNMT-like genes as potential candidate genes associated with both anserine and carnosine. The results presented here will contribute to the ongoing improvement of meat quality to satisfy current consumer demands, which are based on healthier, better-flavored, and higher-quality chicken meat.

Effects of carnosine and histidine-containing dipeptides on biomarkers of inflammation and oxidative stress: a systematic review and meta-analysis

Abstract Context Carnosine and histidine-containing dipeptides (HCDs) are suggested to have anti-inflammatory and antioxidative benefits, but their effects on circulating adipokines and inflammatory and oxidative stress biomarkers remain unclear. Objectives The aim of the present systematic review and meta-analysis was to determine the impact of HCD supplementation on inflammatory and oxidative stress biomarkers. Data Sources A systematic search was performed on Medline via Ovid, Scopus, Embase, ISI Web of Science, and the Cochrane Library databases from inception to 25 January 2023. Data Extraction Using relevant key words, trials investigating the effects of carnosine/HCD supplementation on markers of inflammation and oxidative stress, including C-reactive protein (CRP), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), adiponectin, malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), total antioxidant capacity (TAC), and catalase (CAT) were identified. Meta-analyses were conducted using random-effects models to calculate the weighted mean differences (WMDs) and 95% confidence intervals (CIs). Data Analysis A total of 9 trials comprising 350 participants were included in the present meta-analysis. Carnosine/HCD supplementation led to a significant reduction in CRP (WMD: –0.97 mg/L; 95% CI: –1.59, –0.36), TNF-α (WMD: –3.60 pg/mL; 95% CI: –7.03, –0.18), and MDA (WMD: –0.34 μmol/L; 95% CI: –0.56, –0.12) and an elevation in CAT (WMD: 4.48 U/mL; 95% CI: 2.43, 6.53) compared with placebo. In contrast, carnosine/HCD supplementation had no effect on IL-6, adiponectin, GSH, SOD, and TAC levels. Conclusion Carnosine/HCD supplementation may reduce inflammatory and oxidative stress biomarkers, and potentially modulate the cardiometabolic risks associated with chronic low-grade inflammation and lipid peroxidation. Systematic Review Registration PROSPERO registration no. CRD42017075354.

Histidine-containing dipeptide supplementation improves delayed recall: a systematic review and meta-analysis.

Histidine-containing dipeptides (carnosine, anserine, beta-alanine and others) are found in human muscle tissue and other organs like the brain. Data in rodents and humans indicate that administration of exogenous carnosine improved cognitive performance. However, RCTs results vary. To perform a systematic review and meta-analysis of randomized controlled trials (RCTs) of histidine-containing dipeptide (HCD) supplementation on cognitive performance in humans to assess its utility as a cognitive stabiliser. OVID Medline, Medline, EBM Reviews, Embase, and Cumulative Index to Nursing and Allied Health Literature databases from 1/1/1965 to 1/6/2022 for all RCT of HCDs were searched. 2653 abstracts were screened, identifying 94 full-text articles which were assessed for eligibility. Ten articles reporting the use of HCD supplementation were meta-analysed. The random effects model has been applied using the DerSimonian-Laird method. HCD treatment significantly increased performance on Wechsler Memory Scale (WMS) -2 Delayed recall (Weighted mean difference (WMD) (95% CI (CI)) = 1.5 (0.6, 2.5), P < .01). Treatment with HCDs had no effect on Alzheimer's Disease Assessment Scale-Cognitive (WMD (95% CI) = -0.2 (-1.1, 0.7), P = .65, I2 = 0%), Mini-Mental State Examination (WMD (95% CI) = 0.7 (-0.2, 1.5), P = .14, I2 = 42%), The Wechsler Adult Intelligence Scale (WAIS) Digit span Backward (WMD (95% CI) = 0.1 (-0.3, 0.5), P = .51, I2 = 0%), WAIS digit span Forward (WMD (95% CI) = 0.0 (-0.3, 0.4), P = .85, I2 = 33%) and the WMS-1 Immediate recall (WMD (95% CI) = .7 (-.2, 1.5), P = .11, I2 = 0%). The effect on delayed recall remained in subgroup meta-analysis performed on studies of patients without mild cognitive impairment (MCI), and in those without MCI where average age in the study was above 65. HCD, supplementation improved scores on the Delayed recall examination, a neuropsychological test affected early in Alzheimer's disease. Further studies are needed in people with early cognitive impairment with longer follow-up duration and standardization of carnosine doses to delineate the true effect. PROSPERO registration no. CRD42017075354.

Binding Modes of Carnostatine, Homocarnosine, and Ophidine to Human Carnosinase 1.

Carnosine (CAR), anserine (ANS), homocarnosine (H-CAR), and ophidine (OPH) are histidine-containing dipeptides that show a wide range of therapeutic properties. With their potential physiological effects, these bioactive dipeptides are considered as bioactive food components. However, such dipeptides display low stability due to their rapid degradation by human serum carnosinase 1 (CN1). A dimeric CN1 hydrolyzes such histidine-containing compounds with different degrees of reactivities. A selective CN inhibitor, carnostatine (CARN), was reported to effectively inhibit CN's activity. To date, the binding mechanisms of CAR and ANS have been recently reported, while no clear information about H-CAR, OPH, and CARN binding is available. Thus, in this work, molecular dynamics simulations were employed to elucidate the binding mechanism of H-CAR, OPH, and CARN. Among all, the amine end and imidazole ring are the main players for trapping all of the ligands in a pocket. OPH shows the poorest binding affinity, while CARN displays the tightest binding. Such firm binding is due to the longer amine chain and the additional hydroxyl (-OH) group of CARN. H-CAR and CARN are analogous, but the absence of the -OH moiety in H-CAR significantly enhances its mobility, resulting in the reduction in binding affinity. For OPH which is an ANS analogue, the methylated imidazole ring destroys the OPH-CN1 interaction network at this region, consequentially leading to the poor binding ability. An insight into how CN recognizes and binds its substrates obtained here will be useful for designing an effective strategy to prolong the lifetime of CAR and its analogues after ingestion.

Extensive profiling of histidine-containing dipeptides reveals species- and tissue-specific distribution and metabolism in mice, rats, and humans.

Histidine-containing dipeptides (HCDs) are pleiotropic homeostatic molecules with potent antioxidative and carbonyl quenching properties linked to various inflammatory, metabolic, and neurological diseases, as well as exercise performance. However, the distribution and metabolism of HCDs across tissues and species are still unclear. Using a sensitive UHPLC-MS/MS approach and an optimized quantification method, we performed a systematic and extensive profiling of HCDs in the mouse, rat, and human body (in n = 26, n = 25, and n = 19 tissues, respectively). Our data show that tissue HCD levels are uniquely produced by carnosine synthase (CARNS1), an enzyme that was preferentially expressed by fast-twitch skeletal muscle fibres and brain oligodendrocytes. Cardiac HCD levels are remarkably low compared to other excitable tissues. Carnosine is unstable in human plasma, but is preferentially transported within red blood cells in humans but not rodents. The low abundant carnosine analogue N-acetylcarnosine is the most stable plasma HCD, and is enriched in human skeletal muscles. Here, N-acetylcarnosine is continuously secreted into the circulation, which is further induced by acute exercise in a myokine-like fashion. Collectively, we provide a novel basis to unravel tissue-specific, paracrine, and endocrine roles of HCDs in human health and disease.

Metabolic and microbiota response to arginine supplementation and cyclic heat stress in broiler chickens.

Little attention has been paid to the biological role of arginine and its dietary supplementation in broilers under heat stress (HS) conditions. Therefore, the main aim of this study was to assess the response of broilers to arginine supplementation and cyclic HS, with a focus on liver, pectoral muscle, and blood metabolic profiles and the cecal microbiota. Day-old male Ross 308 broilers (n = 240) were placed in 2 rooms with 12 pens each for a 44-day trial. Pens were assigned to one of two groups (6 pens/group/room): the control group (CON) was given a basal diet in mash form and the treated group (ARG) was fed CON diet supplemented with crystalline L-arginine. The total arginine:lysine ratio of CON diet ranged between 1.02 and 1.07, while that of ARG diet was 1.20. One room was constantly kept at thermoneutral (TN) conditions, while the birds in the other room were kept at TN conditions until D34 and subjected to cyclic HS from D35 onwards (∼34°C; 9:00 A.M.-6:00 P.M.). Blood, liver, Pectoralis major muscle, and cecal content were taken from 2 birds per pen (12 birds/group/room) for metabolomics and microbiota analysis. Growth performance data were also collected on a pen basis. Arginine supplementation failed to reduce the adverse effects of HS on growth performance. Supplemented birds showed increased levels of arginine and creatine in plasma, liver, and P. major and methionine in liver, and reduced levels of glutamine in plasma, liver, and P. major. HS altered bioenergetic processes (increased levels of AMP and reduced levels of fumarate, succinate, and UDP), protein metabolism (increased protein breakdown to supply the liver with amino acids for energy production), and promoted the accumulation of antioxidant and protective molecules (histidine-containing dipeptides, beta-alanine, and choline), especially in P. major. Arginine supplementation may have partially counterbalanced the effects of HS on energy homeostasis by increasing creatine levels and attenuating the increase in AMP levels, particularly in P. major. It also significantly reduced cecal observed diversity, while HS increased alpha diversity indices and affected beta diversity. Results of taxonomic analysis at the phylum and family level are also provided.

Improving the enzymatic activity of l-amino acid α-ligase for imidazole dipeptide production by site-directed mutagenesis.

Imidazole dipeptides, histidine-containing dipeptides, including carnosine (β-alanyl-l-histidine), anserine (β-alanyl-3-methyl-l-histidine), and balenine (β-alanyl-1-methyl-l-histidine) in animal muscles have physiological functions, such as significant antioxidant and antifatigue effects. They are obtained by extraction from natural raw materials, including chicken and fish meat. However, using natural raw materials entails stable supply and mass production limitations. l-amino acid α-ligase (Lal) catalyzes the formation of various dipeptides from unprotected l-amino acids by conjugating with adenosine 5'-triphosphate (ATP) hydrolysis reaction. In this study, site-directed mutagenesis of Lal was applied to establish an efficient method for producing imidazole dipeptides by the enzymatic process. We significantly improved the conversion rate from substrate amino acids compared with wild-type Lal.

Exploring the structural and dynamic differences between human carnosinase I (CN1) and II (CN2).

Human carnosinases (CNs) are dimeric dipeptidases in the metallopeptidase M20 family. Two isoforms of carnosinases (Zn2+ -containing carnosinase 1 (CN1) found in serum and Mn2+ -carnosinase 2 (CN2) in tissue) were identified. Both CNs cleave histidine-containing (Xaa-His) dipeptides such as carnosine where CN2 was found to accept a broader spectrum of substrates. A loss of CN function, resulting in a high carnosine concentration, reduces risk for diabetes and neurological disorders. Although several studies on CN activities and its Michaelis complex were conducted, all shed the light on CN1 activity where the CN2 data is limited. Also, the molecular details on CN1 and CN2 similarity and dissimilarity in structure and function remain unclear. Thus, in this work, molecular dynamics (MD) simulations were employed to study structure and dynamics of human CN1 and CN2 in comparison. The results show that the different catalytic ability of both CNs is due to their pocket size and environment. CN2 can accept a wider range of substrate due to the wider mouth of a binding pocket. The L1 loop seems to play a role in gating activity. Comparing to CN2, CN1 provides more electronegative entrance, more wettability, and higher stability of catalytic metal ion-pair in the active site which allow more efficient water-mediated catalysis. The microscopic understanding obtained here can serve as a basis for CN inhibition strategies resulting in higher carnosine levels and consequently mitigating complications associated with diseases such as diabetes and neurological disorder.

Mechanistic insights on anserine hydrolyzing activities of human carnosinases

Anserine and carnosine represent histidine-containing dipeptides that exert a pluripotent protective effect on human physiology. Anserine is known to protect against oxidative stress in diabetes and cardiovascular diseases. Human carnosinases (CN1 and CN2) are dipeptidases involved in the homeostasis of carnosine. In poikilothermic vertebrates, the anserinase enzyme is responsible for hydrolyzing anserine. However, there is no specific anserine hydrolyzing enzyme present in humans. In this study, we have systematically investigated the anserine hydrolyzing activity of human CN1 and CN2. A targeted multiple reaction monitoring (MRM) based approach was employed for studying the enzyme kinetics of CN1 and CN2 using carnosine and anserine as substrates. Surprisingly, both CN1 and CN2 can hydrolyze anserine effectively. The observed catalytic turnover rate (Vmax/[E]t) was 21.6 s−1 and 2.8 s−1 for CN1 and CN2, respectively. CN1 is almost eight-fold more efficient in hydrolyzing anserine compared to CN2, which is comparable to the efficiency of the carnosine hydrolyzing activity of CN2. The Michaelis constant (Km) value for CN1 (1.96 mM) is almost three-fold lower compared to CN2 (6.33 mM), representing higher substrate affinity for anserine-CN1 interactions. Molecular docking studies showed that anserine binds at the catalytic site of the carnosinases with an affinity similar to carnosine. Overall, the present study elucidated the inherent promiscuity of human carnosinases in hydrolyzing anserine using a sensitive LC-MS/MS approach.

Molecular insights into the binding of carnosine and anserine to human serum carnosinase 1 (CN1)

Carnosine (CAR) and anserine (ANS) are histidine-containing dipeptides that show the therapeutic properties and protective abilities against diabetes and cognitive deficit. Both dipeptides are rich in meat products and have been used as a supplement. However, in humans, both compounds have a short half-life due to the rapid degradation by dizinc carnosinase 1 (CN1) which is a hurdle for its therapeutic application. To date, a comparative study of carnosine- and anserine-CN1 complexes is limited. Thus, in this work, molecular dynamics (MD) simulations were performed to explore the binding of carnosine and anserine to CN1. CN1 comprises 2 chains (Chains A and B). Both monomers are found to work independently and alternatingly. The displacement of Zn2+ pair is found to disrupt the substrate binding. CN1 employs residues from the neighbour chain (H235, T335, and T337) to form the active site. This highlights the importance of a dimer for enzymatic activity. Anserine is more resistant to CN 1 than carnosine because of its bulky and dehydrated imidazole moiety. Although both dipeptides can direct the peptide oxygen to the active Zn2+ which can facilitate the catalytic reaction, the bulky methylated imidazole on anserine promotes various poses that can retard the hydrolytic activity in contrast to carnosine. Anserine is likely to be the temporary competitive inhibitor by retarding the carnosine catabolism.

Histidine-Containing Dipeptides Mitigate Salt Stress in Plants by Scavenging Reactive Carbonyl Species.

Reactive oxygen species (ROS) are critical factors that cause damage in salt-stressed plants, but their mechanisms of action in living cells are largely unknown. We investigated the roles of reactive carbonyl species (RCS), i.e., the lipid peroxide-derived α,β-unsaturated aldehydes and ketones, in plant growth retardation under salt stress. When Arabidopsis thaliana Col-0 seeds were exposed to 100 mM NaCl, germination was delayed and the levels of ROS, RCS, and protein carbonylation in the seedlings were increased. Adding the histidine-containing dipeptides carnosine, N-acetylcarnosine, and anserine, which are reported RCS scavengers, restored the germination speed and suppressed the increases in RCS and protein carbonylation but did not affect the ROS level. Increases in the levels of the RCS acrolein, crotonaldehyde, (E)-2-pentenal, and 4-hydroxy-(E)-2-nonenal were positively correlated with the delay of germination and growth inhibition. These RCS, generated downstream of ROS, are thus primarily responsible for the salt-stress symptoms of plants.

Carnosine synthase deficiency in mice affects protein metabolism in skeletal muscle

Carnosine and anserine are abundant peptides found in the skeletal muscle and nervous system in many vertebrates. Several in vitro and in vivo studies have demonstrate that exogenously administered carnosine improves exercise performance. Furthermore, carnosine is an antioxidant and antifatigue supplement. However, the physiological functions of endogenous carnosine and its related histidine-containing dipeptides in a living organism remain unclear. We aimed to clarify the physiological roles of endogenous carnosine by investigating the characteristics of carnosine synthase gene-deficient mice and the effects of carnosine on skeletal muscle protein metabolism. We discovered that carnosine and anserine were undetectable in the skeletal muscle of carnosine synthase knockout mice. We also quantified protein gene expression and enzyme levels in muscle protein metabolism. Gene and protein levels of the muscle protein synthesizer insulin-like growth factor-1 (IGF-1) and the degrading enzyme cathepsin B were markedly lower in carnosine synthase gene-deficient mice than those in wild-type mice. The amount of 3-methylhistidine (a marker for muscle proteolysis) in forced exercise and the weight of the gastrocnemius muscle were considerably lower in carnosine synthase gene-deficient mice than in wild-type mice. Consequently, we showed that carnosine deficiency affects weight maintenance and protein metabolism in skeletal muscle, suggesting that carnosine regulates skeletal muscle protein metabolism.

Absence of endogenous carnosine synthesis does not increase protein carbonylation and advanced lipoxidation end products in brain, kidney or muscle

Carnosine and other histidine-containing dipeptides are expected to be important anti-oxidants in vertebrates based on various in vitro and in vivo studies with exogenously administered carnosine or its precursor β-alanine. To examine a possible anti-oxidant role of endogenous carnosine, mice lacking carnosine synthase (Carns1−/−) had been generated and were examined further in the present study. Protein carbonylation increased significantly between old (18 months) and aged (24 months) mice in brain and kidney but this was independent of the Carns1 genotype. Lipoxidation end products were not increased in 18-month-old Carns1−/− mice compared to controls. We also found no evidence for compensatory increase of anti-oxidant enzymes in Carns1−/− mice. To explore the effect of carnosine deficiency in a mouse model known to suffer from increased oxidative stress, Carns1 also was deleted in the type II diabetes model Leprdb/db mouse. In line with previous studies, malondialdehyde adducts were elevated in Leprdb/db mouse kidney, but there was no further increase by additional deficiency in Carns1. Furthermore, Leprdb/db mice lacking Carns1 were indistinguishable from conventional Leprdb/db mice with respect to fasting blood glucose and insulin levels. Taken together, Carns1 deficiency appears not to reinforce oxidative stress in old mice and there was no evidence for a compensatory upregulation of anti-oxidant enzymes. We conclude that the significance of the anti-oxidant activity of endogenously synthesized HCDs is limited in mice, suggesting that other functions of HCDs play a more important role.

TAMI-77. CHARACTERIZATION OF METABOLIC DIFFERENCES IN MENINGIOMAS USING TUMOR TISSUE AND PATIENT-DERIVED CELL LINES

Abstract As more information emerges on metabolic changes in brain pathologies, mass spectrometry methodologies are needed to investigate cellular changes in primary brain tumors. Many approaches use technologies that lack sensitivity and selectivity, which hinders discovering potential novel diagnostic and prognostic features. Fresh frozen tissue from 26 patients (57% women, 43% men, age 21-67) underwent surgical resection for newly diagnosed meningiomas. We used patient-derived cell lines and arachnoid cells to validate and compare metabolic differences in patient tumor tissue. Primary metabolism (GC-TOF), lipidomics, and biogenic amines (RPLC and HILIC-HRMS) were analyzed using 10 mg of tissue and 1x106 cells. RESULTS: Primary metabolism and lipidomics showed significant differences between Grade I and Grade II tumors. We identified over one thousand total metabolites through authentic standards and MS2 fragmentation patterns. Ascorbic Acid was enriched in Grade I tumors compared to Grade II and enriched in tumors compared to non-neoplastic dura tissue from the same patient. Lysosomal activation in atypical meningiomas is indicated by a 2-fold increase (compared to Grade I tumors) of Bis(monoacylglycero)phosphate metabolites. We grouped metabolites into fifteen classes based on chemical ontology and function. Statistically significant differences were observed in total amino acids (AA), basic AA, cyclic AA, sulfur-containing AA, branched-chain AA, dipeptides, histidine-containing dipeptides, vitamins and cofactors, glutathione metabolites, acylcarnitine’s, sphingomyelins, phosphatidylethanolamines, phosphatidylinositol’s, cardiolipins, and nucleic acids. Thymine-containing nucleic acids and biogenic monoamines were 2-fold higher in Grade I compared to Grade II tumors. Using novel untargeted metabolomics, we found multiple classes of metabolites that were enriched in Grade II meningiomas compared to Grade I. This points towards possible pathways that drive malignancy and biomarkers that could be useful for diagnosis, grading, and treatment selection.

Skeletal muscle histidine-containing dipeptide contents are increased in freshwater turtles (C. picta bellii) with cold-acclimation

Freshwater turtles found in higher latitudes can experience extreme challenges to acid-base homeostasis while overwintering, due to a combination of cold temperatures along with the potential for environmental hypoxia. Histidine-containing dipeptides (HCDs; carnosine, anserine and balenine) may facilitate pH regulation in response to these challenges, through their role as pH buffers. We measured the HCD content of three tissues (liver, cardiac and skeletal muscle) from the anoxia-tolerant painted turtle (C. picta bellii) acclimated to either 3 or 20 °C. HCDs were detected in all tissues, with the highest content shown in the skeletal muscle. Turtles acclimated to 3 °C had more HCD in their skeletal muscle than those acclimated to 20 °C (carnosine = 20.8 ± 4.5 vs 12.5 ± 5.9 mmol·kg DM−1; ES = 1.59 (95%CI: 0.16–3.00), P = 0.013). The higher HCD content shown in the skeletal muscle of the cold-acclimated turtles suggests a role in acid-base regulation in response to physiological challenges associated with living in the cold, with the increase possibly related to the temperature sensitivity of carnosine's dissociation constant.

Quantification of Histidine-Containing Dipeptides in Dolphin Serum Using a Reversed-Phase Ion-Pair High-Performance Liquid Chromatography Method

The quantification of histidine-containing dipeptides (anserine, carnosine, and balenine) in serum might be a diagnostic tool to assess the health condition of animals. In this study, an existing reversed-phase ion-pair high-performance liquid chromatography (HPLC)–ultraviolet detection method was improved and validated to quantify serum anserine, carnosine, and balenine levels in the dolphin. The serum was deproteinized with trichloroacetic acid and directly injected into the HPLC system. Chromatographic separation of the three histidine-containing dipeptides was achieved on a TSK–gel ODS-80Ts (4.6 mm × 150 mm, 5 µm) analytical column using a mobile phase of 50 mmol/L potassium dihydrogen phosphate (pH 3.4) containing 6 mmol/L 1-heptanesulfonic acid and acetonitrile (96:4). The standard curve ranged from 0.1 µmol/L to 250 µmol/L. The average accuracy of the intra- and inter-analysis of anserine, carnosine, and balenine was 97–106%. The relative standard deviations of total precision (RSDr) of anserine, carnosine, and balenine in dolphin serum were 5.9%, 4.1%, and 2.6%, respectively. The lower limit of quantification of these compounds was 0.11–0.21 µmol/L. These results indicate that the improved method is reliable and concise for the simultaneous determination of anserine, carnosine, and balenine in dolphin serum, and may be useful for evaluation of health conditions in dolphins. Furthermore, this method can also be applied to other biological samples.

Absence Of Histidine Dipeptides Does Not Affect Muscle Contractile Proprieties: Study With Carns1 Knockout Rats

Carnosine and anserine are a histidine-containing dipeptides (HCDs) abundantly expressed in the skeletal muscle. Although several properties have been ascribed to HCDs (e.g., H+ buffering and Ca2+ handling), it remains unknown whether it is relevant for muscle contractility in vivo. PURPOSE: To investigate the impact of the absence of HCDs on muscle contractile properties assessed in vivo. METHODS: We developed a novel CARNS1 knockout (KO) rat strain using the CRISPR/Cas9 technology to generate rats that are fully devoid of HCDs, and thus examine its role on muscle function. Male wild-type (WT) and KO rats (4 months-old) were used. We examined whether CARNS1 KO impacted: i) exercise tolerance in a progressive maximal treadmill exercise test; ii) the in vitro muscle buffering capacity using the titration method; iii) force generation, contractile properties and tolerance to fatigue using the in situ assessment of the tibialis anterioris function evoked by electrical stimulation of the sciatic nerve. Data are presented as mean ± SD. Fatigue test were analysed with mixed models. For all other variables, Welch’s two-sample t-tests for unequal sample sizes were used to compare the groups (RStudio v.4.0.0). RESULTS: Exercise tolerance was similar between KO and WT (KO: n = 6, WT: n = 4; p = 0.34). We next determined the in vitro buffering capacity of skeletal muscle extracts, which confirmed no effect of the absence of HCDs on muscle buffering capacity (n = 15 per group; p = 0.85). The absence of HCDs did not impact muscle resistance to fatigue (KO: n = 10, WT: n = 7; p = 0.37), peak force attained in single twitch and in maximal tetanic contractions, as well as time to peak force and half-relaxation time (KO: n = 10/WT: n = 7; all p > 0.05) (Table). CONCLUSION: HCDs absence did not affect exercise capacity, force generation, fatigability, and the in vitro buffering capacity, indicating that carnosine and anserine are not essential for pH regulation or muscle function. Supported by FAPESP and CAPES

Responses to β-alanine and carnosine supplementation of mussel Mytilus coruscus as revealed by UPLC–MS/MS based untargeted metabolomics

Histidine-containing dipeptides (HCDs) have been demonstrated with important physiological functions in vertebrates. This study was executed to investigate the contents and metabolic profiles of HCDs in an invertebrate, the mussel Mytilus coruscus was selected. The carnosine/anserine analysis indicated that M. coruscus contain anserine with a higher concentration than that of carnosine, and the increases in HCDs concentrations due to β-alanine injection and carnosine injection were observed, confirming that Mytilus belongs to a species with the ability of HCDs synthesis. Further metabolomics analysis was performed based on ultra-performance liquid chromatography-tandem mass spectrometry technology to explore the effects of β-alanine injection and carnosine injection, respectively, on the whole mussel tissue. A total of 1,868 significantly differential metabolites (SDMs) were detected between the β-alanine injected, carnosine injected, and the control M. coruscus groups. The SDMs induced by β-alanine injection were enriched mainly in energy metabolism, lipid metabolism, and amino acid metabolism of mussel, while carnosine injection mainly disrupted arachidonic acid metabolism, steroid hormone biosynthesis, and retinol metabolism in the mussel. The activities of antioxidant enzymes from the whole mussel tissue extracts before and after β-alanine/carnosine injection were measured, and both treatments showed significant elevation of enzymatic activities of superoxide dismutase and catalase. Considering the positive effects of β-alanine injection on mussel metabolism, this study provided a clue for the potential application of β-alanine supplementation in mussel farming.

Histidine dipeptides are key regulators of excitation-contraction coupling in cardiac muscle: Evidence from a novel CARNS1 knockout rat model

Histidine-containing dipeptides (HCDs) are abundantly expressed in striated muscles. Although important properties have been ascribed to HCDs, including H+ buffering, regulation of Ca2+ transients and protection against oxidative stress, it remains unknown whether they play relevant functions in vivo. To investigate the in vivo roles of HCDs, we developed the first carnosine synthase knockout (CARNS1−/−) rat strain to investigate the impact of an absence of HCDs on skeletal and cardiac muscle function. Male wild-type (WT) and knockout rats (4 months-old) were used. Skeletal muscle function was assessed by an exercise tolerance test, contractile function in situ and muscle buffering capacity in vitro. Cardiac function was assessed in vivo by echocardiography and cardiac electrical activity by electrocardiography. Cardiomyocyte contractile function was assessed in isolated cardiomyocytes by measuring sarcomere contractility, along with the determination of Ca2+ transient. Markers of oxidative stress, mitochondrial function and expression of proteins were also evaluated in cardiac muscle. Animals were supplemented with carnosine (1.8% in drinking water for 12 weeks) in an attempt to rescue tissue HCDs levels and function. CARNS1−/− resulted in the complete absence of carnosine and anserine, but it did not affect exercise capacity, skeletal muscle force production, fatigability or buffering capacity in vitro, indicating that these are not essential for pH regulation and function in skeletal muscle. In cardiac muscle, however, CARNS1−/− resulted in a significant impairment of contractile function, which was confirmed both in vivo and ex vivo in isolated sarcomeres. Impaired systolic and diastolic dysfunction were accompanied by reduced intracellular Ca2+ peaks and slowed Ca2+ removal, but not by increased markers of oxidative stress or impaired mitochondrial respiration. No relevant increases in muscle carnosine content were observed after carnosine supplementation. Results show that a primary function of HCDs in cardiac muscle is the regulation of Ca2+ handling and excitation-contraction coupling.